WO2008056759A1

WO2008056759A1 - Method for production of dipeptide

Info

Publication number: WO2008056759A1
Application number: PCT/JP2007/071768
Authority: WO
Inventors: Akihiro Senoo; Kazuhiko Tabata; Makoto Yagasaki; Yoshiyuki Yonetani
Original assignee: Kyowa Hakko Bio Co., Ltd.
Priority date: 2006-11-09
Filing date: 2007-11-09
Publication date: 2008-05-15
Also published as: JPWO2008056759A1

Abstract

A protein having a dipeptide synthetic activity; DNA encoding the protein; recombinant DNA comprising the DNA; a transformant produced by the transformation with the recombinant DNA; a method for producing a protein having a dipeptide synthetic activity by using the transformant or the like; a method for producing a dipeptide by using a protein having a dipeptide synthetic activity; and a method for producing a dipeptide by using a culture of a transformant or microorganism capable of producing a protein having a dipeptide synthetic activity or the like as an enzyme source.

Description

Specification

Dipeptide production method

Technical field

[0001] The present invention relates to a protein having dipeptide synthesis activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant transformed with the recombinant DNA, and a dipeptide synthesis activity The present invention relates to a method for producing a protein, a method for producing a dipeptide using a protein having dipeptide synthesis activity, and a method for producing a dipeptide using a microorganism or a transformant that produces a protein having dipeptide synthesis activity.

Background art

[0002] As a protein having peptide bond-forming activity at the α-carboxyl group of L-amino acid, a bacillicin synthase that is a dipeptide antibiotic derived from a microorganism belonging to the genus Bacillus (see Patent Document 1), Streptomyces' A diketobiperazine synthase derived from Streptomvce salbulus (see Patent Document 2) and a protein derived from Ralstonia's solanacelam (see Patent Document 3) are known! /.

[0003] However, due to the substrate specificity of the enzyme, there are some dipeptides whose production efficiency is not sufficient, and therefore, a new dipeptide synthase having a substrate specificity different from that of the enzyme is required.

Streptococcus mutans (StreiDtococcus mutans) UA159, Streptococcus pneumoniae ATCC BAA-334, Actinenofungus' F ° Norreuni hummonie (Actinobacillus pleuropneumoniaes) The base sequence of chromosomal DNA of the strain and Treponema isnllm ATCC35405 strain is also known to be! / !, and the ORF is estimated for each chromosomal DNA (see Non-Patent Documents 3 to 7). However, the functions of the proteins encoded by Streptococcus mutans SMU1321c, Streptococcus numonie SP0885, Actinobacillus pluronyeumonie Aple02000835, Luminescence Plul218 and Treponema denticola TDE2209 Actually encodes a functional protein V, whether or not! / Wow! /.

Patent Document 1: International Publication No. 2004/058960 Pamphlet

Patent Document 2: Pamphlet of International Publication No. 2005/103260

Patent Document 3: Pamphlet of International Publication No. 2006/101023

Non-Patent Document 1: J. Ind. Microbiol., 2, 201-208 (1987)

Non-Patent Document 2: Enzyme. Microbial. Technol., 29, 400-406 (2001)

^ Patent text d: http://gib.genes.nig.ac.jp/single/index.np?spid=¾mut_UAl 59 Non-patent text 4: http://www.ncbi.nlm.nin.gov/entrez/ query.fcgi? db ⁼ genome & cmd = Ret rieve & dopt = Overview & list—uids = 5281

Non-Patent Document 5: http://www.ncbi.nlm.nin.gov/entrez/ query. Fcgi? Db = genome & cmd = Ret rieve & dopt = Overview & list—uids = 5199

Non-patent literature: http://www.ncbi.nlm.nin.gov/entrez/ query. Fcgi? Db = genome & cmd = Ret rieve & dopt = Overview & list— mds = 047

Non-patent text: http://www.ncbi.nlm.nin.gov/entrez/ query. Fcgi? Db = genome & cmd = Ret rieve & dopt = Overview & list— mds = 08l

Disclosure of the invention

Problems to be solved by the invention

[0004] An object of the present invention is to provide a protein having dipeptide synthesis activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant transformed with the recombinant DNA, the trait A method for producing a protein having dipeptide synthesis activity using a transformant, a method for producing a dipeptide using a protein having dipeptide synthesis activity, and a culture of a transformant or microorganism producing a protein having dipeptide synthesis activity Another object is to provide a method for producing a dipeptide using a product or the like as an enzyme source.

Means for solving the problem

[0005] The present invention relates to the following (1) to (; 10).

(1) The protein according to any one of [1] to [3] below.

[1] SEQ ID NO :; a protein having an amino acid sequence represented by any of 5 to 5

[2] In the amino acid sequence represented by any of SEQ ID NOs: 1 to 5, one or more amino acids are A protein consisting of a deleted, substituted or added amino acid sequence and having dipeptide synthesis activity

[3] A protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by any of SEQ ID NOs: 1 to 5 and having a dipeptide synthesis activity

(2) The DNA according to [1] to [3] below or any one of the following.

[1] DNA encoding the protein of (1) above

[2] DNA having a base sequence represented by any one of SEQ ID NOs: 6 to 10

[3] DNA encoding a protein that hybridizes with DNA having a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 6 to 10 under stringent conditions and has a dipeptide synthesis activity

(3) A recombinant DNA containing the DNA of (2) above.

[0006] (4) A transformant having the recombinant DNA of (3) above.

(5) The transformant according to (4) above, wherein the transformant is obtained by using a microorganism as a host.

(6) The transformant according to (5) above, wherein the microorganism is a fine cattle belonging to the genus Escherichia.

[0007] (7) A microorganism having the ability to produce the protein of (1) above is cultured in a medium, the protein is produced and accumulated in the culture, and the protein is collected from the culture. ) Protein production method.

(8) The production method of (7) above, wherein the microorganism having the ability to produce the protein of (1) is! /, The displacement force, and one transformant of (4) to (6) above.

[0008] (9) A culture of a microorganism having the ability to produce the protein of (1) above or a processed product of the culture, or the protein of (1) above and one or more amino acids in an aqueous medium A method for producing a dipeptide, wherein the dipeptide is produced and accumulated in the medium, and the dipeptide is collected from the medium.

(10) The production method of (9) above, wherein the microorganism having the ability to produce the protein of (1) is the! /, Shear force, or one transformant of (4) to (6) above.

The invention's effect [0009] According to the present invention, a protein having an activity of synthesizing a dipeptide can be produced, and the dipeptide can be produced using the protein or a transformant or a microorganism having an ability to produce the protein.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] 1. Protein of the present invention

As the protein of the present invention,

[1] SEQ ID NO :; a protein having an amino acid sequence represented by any one of! To 5,

[2] a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5 and having dipeptide synthesis activity; and

[3] a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, and having a dipeptide synthesis activity;

Can give.

[0011] In the above, a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added and having dipeptide synthesis activity is Molecular Cloning, A Laboratory Manual, Second Edition (1989), Third Edition. (2001), Cold Spring Harbor Laboratory Press (hereinafter abbreviated as Molecular. Cloning 2nd Edition, 3rd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) 'Molecular' biology), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research , 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc., for example, in any one of SEQ ID NOS: 1-5. By introducing site-specific mutations into the DNA encoding the protein comprising the amino acid sequence The power S to acquire.

[0012] The number of amino acids to be deleted, substituted or added is not particularly limited, but is such a number that can be deleted, substituted or added by a known method such as the above-mentioned site-directed mutagenesis. Several dozen, preferably; !!-20, more preferably 1-; 10, more preferably 1-5. In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, one or more amino acids are deleted, substituted or added. One or more amino acids are deleted at any position in the same sequence, It may be substituted or added.

[0013] The amino acid positions at which amino acids can be deleted or added include, for example, 1 to several amino acids on the N-terminal side and C-terminal side of the amino acid sequence represented by any of SEQ ID NOs: 1 to 5. Use the power S to raise mino acid.

Deletions, substitutions or additions may occur at the same time, and the substituted or added amino acids may be natural or unnatural. Natural amino acids include L-alanin, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-sip Icin, L-lysine, L-aminoreginine, L-methionine, L-phenolanolanine , L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine

[0014] Examples of amino acids that can be substituted with each other are shown below. Amino acids contained in the same group can be substituted for each other.

Group A: leucine, isoleucine, norleucine, valine, norpaline, alanine, 2-aminobutanoic acid, methionine, 0-methylserine, t-butylglycine, t-butylalanine, cyclohexenolealanine

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-amino adipic acid, 2-aminosuberic acid

Group C: Asparagine, glutamine

Group D: lysine, arginine, ornithine, 2,4-dianaminobutanoic acid, 2,3-dianaminopropionic acid

Group E: proline, 3-hydroxyproline, 4-hydroxyproline

Group F: serine, threonine, homoserine

Group G: phenylalanin, tyrosine

In order for the protein of the present invention to have dipeptide synthesis activity, the homology with the amino acid sequence represented by any one of SEQ ID NOs:! To 5 is 80% or more, preferably 90% or more, more preferably 94 % Or more, more preferably 98% or more, particularly preferably 99% or more It is desirable to have a lifetime.

[0015] The homology between amino acid sequences and nucleotide sequences is determined by the algorithm by Karlin and Altschul BLAS T [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990). )]. Based on this algorithm BLAST, programs called BLAST N and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)].

For base sequence analysis by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. When amino acid sequences are analyzed by BLASTX based on BLAST, parameters are, for example, score = 50 and wordlength = 3. Specific methods of these analysis methods are known (http: 〃 WW w.ncbi.nlm.nih.gov.)

[0016] Further, the amino acid sequence represented by any one of SEQ ID NOs:! To 5 is 80% or more, preferably 90% or more, more preferably 94% or more, still more preferably 98% or more, and particularly preferably 99%. A protein comprising the amino acid sequence having the above homology and having dipeptide synthesis activity is also a protein of the present invention. Amino acid sequence homology can be determined using BLAST or FASTA as described above.

[0017] As a means for confirming that the protein of the present invention is a protein having dipeptide synthesis activity, for example, a transformant expressing the protein of the present invention is prepared by using a DNA recombination method, After producing the protein of the present invention using the transformant, the protein of the present invention, one or more amino acids, preferably two amino acids selected from L-amino acid and glycine, and ATP are present in an aqueous medium, A method for analyzing whether or not a dipeptide is produced and accumulated in the aqueous medium by HPLC or the like can be mentioned.

2. DNA of the present invention

As the DNA of the present invention,

[1] DNA encoding the protein of the present invention according to [1] to [3] in 1 above,

[2] DNA having a base sequence represented by any one of SEQ ID NOs: 6 to 10; and

[3] Hybridizes with DNA having a base sequence complementary to the base sequence represented by any of SEQ ID NOs: 6 to 10 under stringent conditions and has dipeptide synthesis activity DNA encoding proteins,

Can give.

[0018] Here, "/," ibridize "is a process in which DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific nucleotide sequence or a part of the DNA sequence is useful as a probe for Northern or Southern blot analysis, or DNA of a length that can be used as an oligonucleotide primer for PCR analysis. There may be. As DNA used as a probe for Northern or Southern blot analysis, DNA of at least 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more can be mentioned, and DNA used as an oligonucleotide primer is at least The ability to raise DNA of 10 bases or more, preferably 15 bases or more, is obtained with S.

[0019] Methods of DNA hybridization experiments are well known, for example, Molecular 'Cloning 2nd Edition, 3rd Edition, Methods for General and Molecular Bacteriolgy, ASM Pres (1994), Immunology methods manual, In the Academic press (Molecular), determine the conditions for hybridization according to many other standard textbooks in addition to 5 self-documents, and conduct experiments with the power S.

[0020] The above stringent conditions include, for example, a DNA-immobilized filter and probe DNA 50% formamide, 5 X SSC (750 mmo / l sodium chloride, 75 mmol / l sodium citrate), 50 mmol After incubation at 42 ° C in a solution containing 1 / l sodium phosphate (pH 7.6), 5 X Denhardt's solution, 10% dextran sulfate, and 20 g / 1 denatured salmon sperm DNA, For example, stringent conditions lower than the force that can increase the conditions for washing the filter in a 0.2 X SSC solution at about 65 ° C can also be used. Stringent conditions can be changed by adjusting the concentration of formamide (lower stringency decreases the stringency), and changing the salt concentration and temperature conditions. Low stringent conditions include, for example, 6 X SSCE (20 X SSCE is 3 mol / l sodium chloride, 0.2 mol / 1 sodium dihydrogen phosphate, 0.02 mol / l EDTA, ρΗ7.4), 0.5 Incubate at 37 ° C in a solution containing 1% SDS, 30% formamide, 100 g / 1 denatured salmon sperm DNA, then 1 X SSC, 0.1% SDS solution at 50 ° C Increase the conditions for cleaning with Power S can be. Further, as a lower stringent condition, the hybridization is performed using a solution having a high salt concentration (for example, 5 × SSC) under the above-mentioned low stringent condition, and then washed. Ability to raise conditions S.

[0021] Various conditions described above can also be set by adding or changing a blocking reagent used for suppressing the background of the hybridization experiment. The addition of the blocking reagents described above may be accompanied by changes in hybridization conditions in order to adapt the conditions.

The DNA that can be hybridized under the above-mentioned stringent conditions includes, for example, at least the base sequence of any of the DNAs described above when calculated based on the above parameters using the above-mentioned programs such as BLAST and FASTA. A DNA having a homology of 80% or more, preferably 90% or more, more preferably 94% or more, still more preferably 98% or more, particularly preferably 99% or more can be mentioned.

[0022] The homology of the base sequence is determined by the force S to be determined using a program such as BLAST or FASTA described above.

The ability to hybridize with the above DNA under stringent conditions S, a DNA encoding a protein having dipeptide synthesis activity means that a recombinant DNA that expresses the DNA is prepared and the recombinant DNA The protein is purified from a culture obtained by culturing a microorganism obtained by introducing a microorganism into a host cell, and the purified protein is used as an enzyme source to produce the enzyme source and one or more amino acids, preferably L- Two kinds of amino acids selected from amino acids and glycine can be present in an aqueous medium, and whether or not dipeptides are generated and accumulated in the aqueous medium can be confirmed by a method of analyzing by HPLC or the like.

3. Microorganisms used in the production method of the present invention and the transformant of the present invention

The microorganism used in the production method of the present invention may be any microorganism that has the ability to produce the protein of the present invention. Examples of the microorganism include Streptococcus genus, Actinobacillus genus, Photolabus ( Examples include microorganisms belonging to the genus Photorhabdus or Treponema, more preferably Streptococcus mutans, Streptococcus pneumoniae ¾ reDtococcus pneumoniae Streptococcus mutans ATCC25175 strain having the ability to produce a protein having the amino acid sequence represented by SEQ ID NO: 1 and represented by SEQ ID NO: 2, more preferably, _TreDone ma denticola. Streptococcus pneumoniae ATCC BAA-334 strain capable of producing a protein having a specific amino acid sequence, Actinobacillus pleuroneumonie strain ATCC27088 capable of producing the protein represented by SEQ ID NO: 3, and represented by SEQ ID NO: 4 And the Trevonema denticola ATCC35405 strain capable of producing the protein having the amino acid sequence represented by SEQ ID NO: 5 and the transformant of the present invention. As a host cell using recombinant DNA containing the DNA of 2 above. And a transformant obtained by transforming the phospholipid by a known method. The host cell may be a bacterium, yeast, animal cell, insect cell or plant cell, preferably a bacterium, more preferably a prokaryotic cell, More preferably, the force S is used to raise fine cattle belonging to the Escherichia moth.

4. Preparation of DNA of the present invention

The DNA of the present invention is, for example, a Southern hybrid to a chromosomal chromosomal DNA library using a probe or primer DNA that can be designed based on the nucleotide sequence represented by any one of SEQ ID NOs: 6 to 10 It can be obtained by PCR using PCR or chromosomal DNA of microorganisms as a cage [PCR Protocols, Academic Press (1990)]. As the above-mentioned Southern hybridization chromosomal DNA library or PCR variant,

(1) In order to obtain a DNA having the base sequence represented by SEQ ID NO: 6, a microorganism belonging to the genus Streptococcus, preferably a microorganism belonging to Streptococcus mutans, more preferably Streptococcus mutans ATCC25175,

(2) In order to obtain a DNA having the base sequence represented by SEQ ID NO: 7, a microorganism belonging to the genus Streptococcus, preferably a microorganism belonging to Streptococcus pneumoniae, more preferably a Streptococcus pneumoniae ATCC BAA-334, (3) In order to obtain a DNA having the base sequence represented by SEQ ID NO: 8, a microorganism belonging to the genus Actinobacillus, preferably a microorganism belonging to Actinobacillus pluronii monnier, more preferably, Actinobacillus plutonimonii ATCC27088

(4) In order to obtain a DNA having the base sequence represented by SEQ ID NO: 9, a microorganism belonging to the genus Photolabus, preferably a microorganism belonging to Photolabs 'Luminescence, more preferably Photolabus' Minescence DSM15139, and

(5) In order to obtain a DNA having the base sequence represented by SEQ ID NO: 10, a microorganism belonging to the genus Treponema, preferably a microorganism belonging to Treponone denticola, more preferably a chromosomal DNA of Treponone denticola ATCC35405 Can be used.

[0024] The DNA base sequence encoding the amino acid sequence represented by any one of SEQ ID NO: 15 with respect to various gene sequence databases is 85% or more, preferably 90% or more, more preferably 95% or more. More preferably, a sequence having a homology of 98% or more, particularly preferably 99% or more is searched, and based on the base sequence obtained by the search, the above-described chromosomal DNA cDNA library of an organism having the base sequence is used. The DNA of the present invention or the DNA used in the production method of the present invention can also be obtained by the method described above.

[0025] The obtained DNA is cut as it is or with an appropriate restriction enzyme and incorporated into a vector by a conventional method, and the resulting recombinant DNA is introduced into a host cell, followed by analysis of a commonly used base sequence. Analysis using a base sequence analyzer such as the dideoxy method [Proc. Natl. Acad. ScL, USA, 74, 5463 (197 7)] or ABI3700 DNA analyzer (Applied Biosystems). DNA sequence can be determined

[0026] When the obtained DNA has a partial length as a result of determining the base sequence, the partial length D

The ability to obtain full-length DNA can be obtained by the Southern hybridization method for chromosomal DNA libraries using NA as a probe.

Furthermore, based on the determined DNA base sequence, the target DN can be obtained by chemically synthesizing using Applied Biosystems 3400 DNA synthesizer etc. manufactured by Applied Biosystems.

A can also be prepared.

[0027] The DNA obtained as described above is represented by any one of SEQ ID NOs: 6 to 10 for example. The ability to raise DNA with IJ

Examples of vectors that incorporate the DNA of the present invention include pBluescriptll KS (+) (Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], pCR—Script Amp SK (+) (Stratagene) ), PT7Blue (Novagene), pCR II (Invitrogen) and pCR-TRAP (Gene Norter).

[0028] Examples of host cells include microorganisms belonging to the genus Escherichia. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli XLl-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli ATCC 12435, Escherichia coli. W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli Νο49 ME8415 etc. can be mentioned.

[0029] As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. ScL, USA 69, 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), electrovolution method [Nucleic Acids Res., 16, 6127 (1988)] and the like.

As the transformant of the present invention obtained by the above method, for example, Escherichia coli BL21, which is a fine cattle having a recombinant DNA containing a DNA having a base sequence represented by any of SEQ ID NOs: 6 to 10, (DE3) / DSMU1321C. BL21 (DE3) / pSP0885, BL21 (Yes.

5. Method for producing the transformant of the present invention

Based on the DNA of the present invention, if necessary, a DNA fragment having an appropriate length containing a portion encoding the protein of the present invention is prepared. In addition, a transformant with an improved production rate of the protein can be obtained by substituting the base in the base sequence of the protein-encoding portion so as to be an optimal codon for host expression.

[0030] A recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector. A transformant producing the protein of the present invention can be obtained by introducing the recombinant DNA into a host cell suitable for the expression vector.

As host cells, bacteria, yeast, animal cells, insect cells, plant cells, etc. that can express the gene of interest can be used.

[0031] As the expression vector, a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed is used.

When a prokaryotic organism such as a bacterium is used as a host cell, the recombinant DNA having the DNA of the present invention can replicate autonomously in the prokaryotic organism, and at the same time, a promoter, a ribosome binding sequence, the DNA of the present invention, a transcription It is preferably a recombinant DNA composed of termination sequences! Contains the genes that control the promoter!

[0032] As expression vectors, pColdl (manufactured by Takara Bio Inc.), pCDF-lb, pRSF-lb (V, also from Novagen Inc.), pMAL-c2x (manufactured by New England Biolabs), pGEX-4T- l (Gee Health Care Bioscience), pTrcHis (Invitrogen), pSE280 (Invitrogen), pGEMEX- ((Promega), pQE-30 (Qiagen), pET_3 (Novagen) 1)? 10 (JP 58-110600), 1? 200 [eight _{8 Biol. Chem., 48,} 669 (1984)], pLSAl [Agric. Biol. Chem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescriptll SK), pBluescript II KS (-) (Stratagene), pTrS30 (prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], TrS3 2 (Escherichia coli JM109 / pTrS32 ( FERM BP-5408), PAC31 (W098 / 123 43), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Bio), pUC118 (Takara Bio), pPAl Sho 63-233798) and the like.

[0033] The promoter may be any as long as it functions in a host cell such as Escherichia coli. For example, promoter (P), 1 ^ promoter (p), P

tro lac L mouth motor, p promoter, p promoter, etc.

R SE

Promote promoter, SPOl promoter, SP02 promoter, penP promoter, etc. In addition, two P promoters in series, tac promoter, lacT7

trp

Mouth motors, artificially designed and modified promoters such as _let I promoter Being the power S

[0034] In addition, the xylA promoter for expression in microorganisms belonging to the genus Bacillus [Appl.

Microbiol. Biotechnol., 35, 594-599 (1991) P54-6 promoter for expression in microorganisms belonging to the genus Corvnebacterium [Appl. Microbiol. Biote chnol., 53, 674-679 (2000)] can also be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (eg, 6 to 18 bases).

[0035] In the recombinant DNA in which the DNA of the present invention is bound to an expression vector, a transcription termination sequence is not necessarily required !, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

Examples of such recombinant DNA include pSMU1321c, pSP0885, pAple0835, pPlul 218 and pTDE2209.

Prokaryotes include Escherichia, Serratia, Bacillus, Brevibacterium 鼠, Corvnebacterium 鼠, Microbacterium, Pseudomonas genus, Agronocterium. (Agrobacterium), Alicyclobacinolus (Alicvclobacillus), Anabena, Anacvstis, Arthrobacter, Azotobacter, Chromatium , Genus Erwinia, genus Methvlobacterium, genus Phormidium, genus Rhodobact, genus Rhodoipseudomonas, genus Rhodosipirillum

Microorganisms belonging to the genus (Svnechoccus), Zvmomonas, etc., for example, Escherichia coli XLl—Blue, Escherichia 'Colli XL2-Blue, Escherichia coli DH 1, Escherichia coli • DH5a, Escherichia coli MC 1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB 101, Escherichia coli Νο · 49, Escherichia coli K3110, Escherichia coli NY49, Escherichia coli MP347, Escherichia 522 'Cori BL2 U Bacillus subtilis ATC C33712, Bacillus' Megaterium ', Brevibaterium' Ammoni Agenes (Brevibacterium ammonia enes), pu, Levinocterium 'Imariofinolem (CC), Brevibacterium saccharolvticum ATCC14066, Brevibacterium. Flavum (Brevibacteri um ATCC67) Revibacterium lac tofermentum ATCC13869, Corvnebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14297, Corynebacterium ammoni-ammoniaDhilum

marcesce ns), Pseudomonas SD. D_0110, Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi ana Bena Syrin force (Anabaena cylindrica), Anabaena doliolum, Anabaena

-aguaej, Arthrobacter, aurescens, er

Grop 'Phonoremis (Arthrobacter globformis), Arthrobacter mvsorens, Arthrobacter mvsorens, Arthrobacter hvdrocarbogiutamicus ineus), Art Hrobacter orotoDhormiae, Arthrobacter or optoffinus, and Arthurobacter

romatium vinosum, Chromatium warmingii, Chromatium fluviatile, Eureviurea, Erwinia uredovora, Erwinia carotovora, Erwinia carotovora, Erwinia carotovora, ananas), Erwinia herbicola, Erwinia Dunctata, Erwinia terreus, Methylobac terium rhodesianum, Methylobacterium extor guens, Phormidium sp. ATCC29409, Rhopa ector Eroides), Rhodopseudomonas blastica, Rhodoipseudomonas marina, Rhodo Dseudomonas palustris, Mouth spirum. Rhodospirillum salexigens), Rhodospi rillum salinarum, Stofftomyces. Ambofaciens, Streptomyces aureofaciens, Streptomyces reptomvces aureus), Streptomyces' Sunga stigma (S ^ eptomvces fungicidicus), Sleff. Tomyces. Streptomvces tanashiensis, Stmotomis vinashius, St. Tomomis vinaceus Power S can be.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the above host cell. For example, a method using calcium ions [Proc. Natl. Acad. ScL, USA, 69, 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), electrovolution method [Nucleic Acids Res., 16, 6127 (1988)] and the like.

When a yeast strain is used as a host cell, for example, YEpl3 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419) or the like can be used as an expression vector. Any promoter that functions in a yeast strain can be used. For example, PH05 promoter, PGK promoter, GAP promoter, ADH promoter motor, _gal 1 promoter, _{gal 10} promoter, heat shock polypeptide promoter And promoters such as MF a 1 promoter and CUP 1 promoter.

Host cells include the genus Saccharomvces, izosaccharomvces, genus Kluvveromvces, genus trichosporon, genus Schioniomvces and genus Pichia. Or yeast strains belonging to the genus Canidaida, such as Saccharomvces cerevisiae, Schizosaccharomyces pombe, Kluvveromvces lactis, Examples include Trichosporon pullulans, Siniomyces' Albius (Schw anniomvces alluviusJ, Pichia pastoris), Cantita utilis (£ andida utilis).

[0038] As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into yeast. For example, the electoral position method [Methods Enzymol., 194, 182 (1990)], Examples include the spheroplast method [Proc. Natl. Acad. ScL, USA, 81, 4889 (1984)] and the lithium acetate method [J. Bacteriol., 153, 163 (1983)].

When animal cells are used as hosts, examples of expression vectors include pcDNAI, pcD M8 (sold by Funakoshi), pAGE107 (Japanese Patent Laid-Open No. 3-22979), pAS3_3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochem, 101, 1307 (1987)], pAGE210, pAMo, p AMoA, etc. it can.

[0039] Any promoter can be used as long as it functions in animal cells. For example, a promoter of the cytomegalovirus (CMV) IE (immediate early) gene, an early promoter of SV40, or a metamouth Examples include thionein promoter, retrowinore promoter, heat shock promoter, SRa promoter and the like. In addition, an enhancer of the IE gene of human CMV may be used together with a promoter.

[0040] As host cells, mouse myeloma cells, rat myeloma cells, mouse cells, hybridoma cells, human cells such as Namalwa cells or Namalva KJM-1 cells, human fetal kidney cells, human leukemia Cells, African green monkey kidney cells, Chinese 'ham CHO cells, which are star cells, HBT5637 (Japanese Patent Laid-Open No. 63-299), and the like. Mouse: SP2 / 0, NSO, etc. for rat myeloma cells, YB2 / 0, etc. for rat myeloma cells, HEK293 (ATCC CRL_1573) for human fetal kidney cells, BALL-1, etc. for human leukemia cells, African green monkey kidney Examples of cells include COS_l and COS-7.

[0041] As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into animal cells. For example, the electopore position method [Cytotechnology, 3, 133 (1990)], Examples include the methods described in the calcium phosphate method (JP-A-2-27075), the ribofusion method [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)], Virology, 52, 456 (1973). it can.

[0042] When insect cells are used as a host, for example, Baculovirus Expression Vectors, A Laboratory Manual, W. H. rreeman and Shim ompany, New York (1992), Karen Kaoru * Protocorores in. Molecular 'Neurology 1 Proteins can be produced by the methods described in Molecular Biology, A Laboratory Manual, Bio / Technology, 6, 47 (1988).

[0043] That is, a recombinant gene transfer vector and a baculovirus are co-introduced into insect cells to obtain a recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to produce a protein. You can make it S.

Examples of the gene transfer vector used in the method include pVL1392, pVLl 393 (manufactured by Orbigen), pBlueBacIII (V, both of which are manufactured by Invitrogen), and the like.

[0044] As a baculovirus, for example, the autographa californica nu clear polyhedrosis virus; which is a virus that infects night stealing insects, can be used with Autographa californica nu clear polyhedrosis virus; Yeah.

Insect cells include Spodoptera frugiperda ovary cells, Trichoplusia πί ovary cells, and cultured cells derived from silkworm ovary.

[0045] Spodoptera fulgiverda ovary cells include Si, Si21 (Baculovirus The culture cells derived from silkworm ovary such as High 5, 、 -ΤΝ-5Β1-4 (manufactured by Invitrogen), etc. mori) N4 etc.

Examples of a method for co-introducing the above recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-227075), the lipofuxion method [Proc. Natl. Acad Sci., USA, 84, 7413 (1987)].

[0046] When plant cells are used as host cells, examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors.

Any promoter can be used as long as it functions in plant cells. Examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter, the rice actin 1 promoter, and the like.

[0047] Examples of host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, oilseed rape, anolefa norefa, rice, wheat and barley.

As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, a method using Agrobacterium (JP 59-140885, JP SHO 60-70080, WO94 / 00977), the Elect Mouth Position Method (JP 60-251887), the method using a particle gun (Gene Gun) (Patent No. 2606856, Japanese Patent No. 2517813), and the like.

6. Method for producing the protein of the present invention

The protein of the present invention can be produced by culturing the microorganism or transformant of the above 3 in a medium, producing and accumulating the protein of the present invention in the culture, and collecting the protein from the culture.

Yes

[0048] The host of the transformant for producing the protein of the present invention may be any of bacteria, yeast, animal cells, insect cells, plant cells, etc., preferably bacteria, more preferably Examples include microorganisms belonging to the genus Escherichia, more preferably microorganisms belonging to Escherichia coli.

The protein of the present invention is expressed using yeast, animal cells, insect cells or plant cells as hosts. In this case, a protein to which a sugar or a sugar chain is added can be obtained.

[0049] The method of culturing the microorganism or the transformant in a medium can be performed according to a conventional method used for culturing the microorganism or the host.

As a medium for culturing a transformant obtained by using a prokaryote such as the above microorganism 3, Escherichia coli or a eukaryote such as yeast as a host, a carbon source, a nitrogen source, and an inorganic salt that can be assimilated by the organism. Any of a natural medium and a synthetic medium may be used as long as it is a medium that can efficiently cultivate transformants.

[0050] As the carbon source, as long as the organism can assimilate, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid, etc. Organic acids, alcohols such as ethanol, propanol and the like can be used.

Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogenous compounds, peptone, meat extract, yeast extract, corns. Chiplicers, casein hydrolysates, soybean meal and soybean meal hydrolysates, various fermented cells, and digested products thereof can be used.

[0051] As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

The culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is 15-40 ° C. The culture time is usually 5-7 days. During culture, pH is 3.0-1

1.0, preferably 5 · 0 to 9 · 0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0052] Further, an antibiotic such as ampicillin or tetracycline may be added to the medium as needed during the culture.

When cultivating a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter. For example, isopropyl 13D thiogalatatopyranoside or the like, and indoleacrylic acid or the like may be added to the medium when cultivating a microorganism transformed with an expression vector using a promoter.

[0053] As a medium for culturing a transformant obtained using animal cells as a host, a commonly used RPMI1640 medium. Am. Med. Assoc., 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], 199 medium [Pro Soc. Biol. Med., 73, 1 (1950)] A medium supplemented with fetal serum or the like can be used.

[0054] Culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C, and the presence of 5% CO.

In addition, antibiotics such as kanamycin, penicillin, streptomycin, etc. may be added to the medium as needed during culture! / ヽ.

As a medium for culturing transformants obtained using insect cells as hosts, commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies) ExCell400, ExCell405 [both manufactured by JRH Biosciences], Grace's Insect Medium [Nature, 195, 788 (1962)] and the like can be used.

[0055] The culture is usually carried out under conditions of pH 6-7, 25-30 ° C, etc. for 1-5 days.

In addition, antibiotics such as gentamicin may be added to the medium as needed during the culture.Transformants obtained using plant cells as hosts are cultured as cells or differentiated into plant cells and organs. can do. As a medium for cultivating the transformant, commonly used Murashige & 'Stag (MS) medium, White medium, or plant hormones such as auxin and cytokinin were added to these mediums. You can use medium etc.

[0056] Cultivation is usually carried out under conditions of pH 5-9 and 20-40 ° C for 3-60 days.

In addition, antibiotics such as kanamycin and hygromycin may be added to the medium as needed during culture.

The method for producing the protein of the present invention includes a method for producing in the host cell, a method for secreting it outside the host cell, and a method for producing it on the outer membrane of the host cell. Depending on the law, the structure of the protein to be produced can be changed.

[0057] When the protein of the present invention is produced in the host cell or on the outer membrane of the host cell, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Roh et al. [Proc Natl. Acad.

Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or JP 05-336

By applying the method described in 963, WO94 / 23021, etc., the protein can be actively secreted outside the host cell.

[0058] That is, by using a gene recombination technique, a protein containing the active site of the protein of the present invention is produced in a form in which a signal peptide is added in front of the protein, so that the protein is actively released outside the host cell. Can be secreted.

Further, according to the method described in JP-A-2-27075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.

[0059] As a method for isolating and purifying the protein of the present invention produced using the transformant producing the protein of the present invention, usual enzyme isolation and purification methods can be used.

For example, when the protein of the present invention is produced in a dissolved state in cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a manton. Crush the cells with a Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract.

[0060] From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation using an organic solvent, etc. Method, anion exchange chromatography using resin such as dimethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), resin such as SS mark harose FF (Falmasia) Cation exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing A purified sample can be obtained by using an electrophoresis method such as electrophoresis alone or in combination.

[0061] When the protein is produced in the form of an insoluble substance in the cell, the cell is similarly collected, disrupted, and centrifuged from the precipitate fraction obtained by a conventional method. The protein After recovering the white matter, the insoluble material of the protein is solubilized with a protein denaturant.

The solubilized solution is diluted with a solution containing no protein denaturing agent or a dilute solution so that the concentration of the protein denaturing agent does not denature the protein, or dialyzed to form the protein into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method as described above.

[0062] When a derivative of the protein of the present invention or a modified sugar thereof is secreted outside the cell, the derivative of the protein or a sugar adduct thereof can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. You can get power S.

[0063] Examples of the protein thus obtained include a protein having an amino acid sequence represented by any one of SEQ ID NOs:! -5.

In addition, the protein of the present invention can be produced as a fusion protein with another protein and purified using affinity chromatography using a substance having an affinity for the fused protein. For example, the method described in Law et al. [Proc. Natl. Acad. ScL, USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], JP-A-5-336963, WO94 / 23021 Similarly, the protein of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

[0064] Also, affinity chromatography using the anti-Flag antibody produced by producing the protein of the present invention as a fusion protein with Flag peptide [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], and can also be purified by the affinity mouth matrix using a metal coordination resin that is produced as a fusion protein with polyhistidine and has a high affinity for polyhistidine. it can. Furthermore, it can also be purified by affinity chromatography using an antibody against the protein itself.

[0065] Based on the amino acid sequence information of the protein obtained above, the protein of the present invention is produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (tbutyloxycarbonyl method). can do. In addition, chemical synthesis may be performed using peptide synthesizers such as Advanced ChemTech, Monokino * enoremer f, Pharmacia, Protein Technology Instrument, Syntheceil-Vega, PerS India, Shimadzu Corporation. it can.

7. Process for producing the dipeptide of the present invention

A culture of the microorganism or transformant described above or a processed product of the culture, or the protein of the present invention described above and one or more amino acids are present in an aqueous medium to produce a dipeptide in the medium. The dipeptide can be produced by accumulating and collecting the dipeptide from the medium.

(1) A method for producing leupeptide using the protein of the present invention as an enzyme source

In the production method of the present invention, when the protein of the present invention is used as an enzyme source, L-amino acid, glycine (Gly) and / 3-alanine (/ 3-- Any amino acid may be used in any combination as long as it is an amino acid selected from the group consisting of Ala). Examples of L-amino acids include L-alanine (L-Ala), L-daltamine (L_Gln), L-gnoretamic acid (L_Glu), L-norine (L-Val), L-leucine (L_Leu), L -Isoleucine (L-Ile), L-proline (L_Pro), L-phenylalanine (L_Phe), L-tryptophan (L-Trp), L-methionine (L-Met), L-serine (L_Ser), L-threonine (L_Thr), L-cysteine (L-Cys), L-asparagine (L-Asn), L_tyrosine (L_Tyr), L_lysine (L-Lys), L-arginine (L-Arg) , L-histidine (L-His), L-asparagine (L- Asp), L-ornithine (L-Om) and L-citrulline (L-Cit).

[0066] As a preferred amino acid to be used in the above production method, when a protein having the amino acid sequence represented by SEQ ID NO: 1 is used as an enzyme source, a combination of the following group H amino acids, an amino acid sequence represented by SEQ ID NO: 2 When using a protein having an amino acid sequence as an enzyme source, the following group I amino acid combinations are used. When using a protein having the amino acid sequence represented by SEQ ID NO: 3 as an enzyme source, the following group J amino acid combinations are used: When a protein having the amino acid sequence shown below is used as the enzyme source, a combination of the following group K amino acids is used. When a protein having the amino acid sequence shown by SEQ ID NO: 5 is used as the enzyme source, You can give a combination.

[0067] Group H: one amino acid selected from L-Phe, L_Tyr, L_Trp, and L_His, L_Ala, L_Se r, _Thr, _Cys, Shiichi Val, then eu, then Ile, _Met , Then _Pro, then _Phe, then _Tyr, then _Trp, then one A sp, L-Glu, L-Asn, L_Gln, L_His, L-Lys, L_Arg, L_0m, L_Cit, Gly and / 3 -Ala One amino acid combination selected from L-Leu and L-Cys, preferably L_Phe and L—Ala, L_Ser, L_Thr, L-Cys, L—Val, L-Leu, L_Ile, L- L-Tyr in combination with one amino acid selected from Met, L_Phe, L—Tyr, L—Trp, L—Gln, L—His, L—Lys, L—Arg, Gly and / 3—Ala And L_Ala, L_Ser, L_Thr, L_Cys and a combination of one amino acid selected from Gly, L-Trp and L-Ala, L-Ser, L-Thr, L-Cys, L-Val, L-Leu, L -Ile, L-Met, L—His, L—Lys, L_Arg, Gly and / 3—Ala. Combined with one amino acid, L—His and L—Ala, L—Ser, L — Thr, L-Cys, L—Met, L—Pro, L—Gln, L—His, L-Lys, L-Arg, Gly and / 3-Ala A combination of L-Cys and L-Leu, more preferably L_Phe and L_Val, L-Leu, L_Ile, L Combination with one amino acid selected from -His, L-Lys and L-Arg

Group I: One amino acid selected from L_Phe, L-Tyr and L-Trp and L_Ala, L_Ser, L_Thr, L-Cys, L_Val, L-Leu, L-Ile, L-Met, L_Pro, L_Phe, L- With one amino acid selected from Tyr, L-Trp, L_Asp, L_Glu, L-Asn, L-Gln, L-His, L-Lys, L-Arg, L_Om, L_Cit, Gly and / 3-Ala Combination, combination of L-Leu and L-Ala, L_Ser, L-Thr, L-Glu, L_Asn, L-Gln, L-His, L-Lys, L_Arg and Gly In combination with L-Pro and one amino acid selected from L-His, L-Lys and L-Arg, preferably L_Phe and L-Ala, L_Ser, L_Thr, L-Cys, L_Phe, L-Tyr L_Trp and L_Ala, L-Ser, L—Thr, L-Cys, L-His, L, L-Trp, L-His, L-Lys, L-Arg and Gly -Lys, L-Arg, Gly and / 3--Ala in combination with one amino acid selected from L-Leu and L-Ala, L_Ser, L_Thr and Gly A combination of L-Pro and L-His, more preferably L-Phe or L-Trp, and one amino acid selected from L_His, L-Lys and L-Arg Combination of L-Pro and L-His

Group J: One amino acid selected from L-Leu, L_Met, L-Phe, L-Tyr, L_Trp and L_His and L—Ala, L-Ser, L_Thr, L-Cys, L_Val, L-Leu, L_Ile, L-Met, L_Pro, L_Phe, L-Tyr, L-Tr L_Asp, L_Glu, L_Asn, L_Gln, L-His L-Lys L-Arg L_Orn, L_Cit, Gly and / 3-Ala And a combination of L-Cys with one amino acid selected from L-Lys, L_His and L-Arg, preferably L_Leu and L-A1 Combination with one amino acid selected from a, L-Ser, L-Thr, L_Cys, L-Met, L_Phe, L_Tyr, L_Trp, L_Glu, L_Asn, L_Gln, L-Cit, Gly and / 3 -Ala, L_Met L_Ala, L_ Ser, L-Thr, L-Cys, L-Val, L-Leu, L_Ile, L-Met, L_Phe, L_Tyr, L-Trp, L_Gln, L-His, L-Lys, L-Arg, L_Phe or L_Trp and L—Ala, L_Ser, L-Thr, L_Cys, L—Val, L-Leu, L_Ile, L-Met in combination with one amino acid selected from L_Cit, Gly and / 3-Ala , L—Phe, L—Tyr, L—Trp, L—Asp, L—Asn, L—Gln, L—His, L—Lys, L-Arg, L—Cit, Gly and / 3 -Ala Combination with one amino acid, combination with one amino acid selected from L_Tyr and L_Ala, L_Ser, L-Thr, L_Cys, L_His, L_Lys, L_Arg and Gly, and L_His with L-Ala, L_Ser, L- Thr, L_Cys, L—Val, L-Leu, L_Ile, L-Asn, L—Gln, L—His, L—Lys, L-Arg, L—Cit, Gly The combination of a combination of one amino acid selected from pre / 3-Ala, more preferably L-Phe or L-Tyr and L-Val, and one amino acid Bareru selected from L_Leu and L_Ile

Group K: L-Phe, L-Trp or L-Tyr and L-Phe, L-Tyr, L_Trp, L_Asp and L_Gln, one amino acid combination, and L_Gln and L_Phe, L-Trp, L- A combination with one amino acid selected from Tyr, L_Glu, L_His, L-Lys and L-Arg, preferably one amino acid selected from L_Gin and L-Phe, L-Trp, L_Tyr and L-Glu A combination of L-Phe or L-Tyr and L_Gln, and a combination of L-Glu and L_Gln

L group: Gly or L-Ala and L-Met, L_Pro, L_Phe, L_Tyr and L_Trp in combination with one amino acid, L-Ser and L-Gln or L-Om in combination, L_Thr and LG lu L-Cys and L_Cys, L-Val, L-Leu, L_Ile, L-Met, in combination with one amino acid selected from L-His, L-Lys, L-Arg, L_Cit and / 3-Ala One amino acid selected from L_Pro, L_Phe, L-Tyr, L-Trp, L-Asp, L-Glu, L-Asn, L-Gln, L-His, L-Lys, L_Arg and / 3-Ala A combination of L_Glu and L-Val, a combination of one amino acid selected from L_Leu and L_Ile, a yarn of L-Ile and L_Phe, one amino acid selected from L-Tyr, L_Trp and L_Cit A combination of L_Pro and L_Phe, L-Tyr, L-Trp, L_His, L-Lys, L-Arg, L-Orn, L-Cit and / 3-Ala. , L_Tyr (Also—Trp and _Phe, then _Tyr, then _Trp, then Asp, then _Gln And _Fiis, teeth - to ys you Bikokorozashi one Argj: Ri]! A combination of one amino acid selected from L-Gln and L-His, a combination of one amino acid selected from L_Lys and L_Arg, 3-Ala only, and L-Cit only, preferably Gly or L- Combination of Ala and L-Pro, combination of L_Cys and L-Glu, and combination of L_Pro and one amino acid selected from L-His, L-Lys and L-Arg, more preferably L-Pro Combination with one amino acid selected from L-His, L-Lys and L-Arg

In the above production method, the protein of the present invention is added in an amount of 0.01 to 100 mg, preferably 0.1 to 10 mg per mg of amino acid used as a substrate.

[0068] In the above production method, the amino acid used as a substrate is 0.1 to 500 g / L, preferably 0.2 to

Add to the aqueous medium for the first time or during the reaction to a concentration of 200 g / L.

In the above production method, ATP can be used as an energy source, and ATP is used at a concentration of 0.5 mmol to 10 mol / L.

The aqueous medium used in the above production method may be an aqueous medium of any component and composition as long as it does not inhibit the dipeptide formation reaction. For example, water, phosphate, carbonate, acetate, Examples thereof include buffers such as borate, citrate, and tris. It also contains alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetoamide.

[0069] The dipeptide formation reaction is carried out in an aqueous medium at pH 5 to ll, preferably pH 6 to 10, 20 to 50 ° C, preferably 25 to 45 ° C, for 2 to 150 hours, preferably 6 to 120 hours. .

Dipeptides produced by the above method include L-Ala, L-Ser, L_Thr, L_Cys, L_Val, L—Leu, L—Ile, L—Met, L—Pro, L—Phe, L—Tyr, L— One or two amino acids selected from Trp, L—Asp, L—Glu, L—Asn, L—Gln, L_His, L-Lys, L-Arg, L_Orn, L_Cit, Gly and / 3-Ala When a dipeptide linked by a peptide bond, preferably a protein having the amino acid sequence represented by SEQ ID NO: 1 is used as the enzyme source, the following M group dipeptides, a protein having the amino acid sequence represented by SEQ ID NO: 2 are used as the enzyme source When using as the enzyme source, the following group N dipeptide, and when using the protein having the amino acid sequence represented by SEQ ID NO: 3 as the enzyme source, the following group 0 dipeptide, the protein having the amino acid sequence represented by SEQ ID NO: 4 as the enzyme When used as a source, the following P group dipeptide, SEQ ID NO: 5 When a protein having an amino acid sequence represented by the following is used as an enzyme source: A dipeptide in which a species or two amino acids are linked by a peptide bond can be mentioned. Group M: One amino acid selected from L-Phe, L_Tyr, L_Trp and L_His, L_Ala, L_Ser, then _Thr, then _Cys, then val, then eu, then _Ile, then _Met, then _Pro, _Phe, _Tyr, _Trp, ichiichi A sp, L-Glu, L-Asn, L_Gln, L_His, L-Lys, L_Arg, L_Orn, L_Cit, Gly and / 3 -Ala A dipeptide consisting of L-Leu and L-Cys, preferably L_Phe and L-Ala, L_Ser, L_Thr, L_Cys, L-Val, L-Leu, L_Ile, L-Met, L-Phe, L-Tyr, L_Trp, L_Gln, L_His, L-Lys, L_Arg, Gly and / 3-Ala selected from one dipeptide consisting of L-Tyr and L_Ala, L_Ser, L_Thr, L_Cys and Gly Dipeptide consisting of one amino acid, L-Trp and L-Ala, L_Ser, L-Thr, L-Cys, L-VaU L-Leu, L_Ile, L_Met, L_His, L_Lys, L-Arg, Gly and / 3 One amino acid selected from -Ala L-His and L-Ala, L_Ser, L-Thr, L_Cys, L—Met, L—Pro, L—Gln, L-His, L—Lys, L—Arg, Gly and / 3—Ala A dipeptide consisting of one amino acid selected from the above, and a dipeptide consisting of L-Cys and L-Leu, more preferably L-Phe and L-Val, L-Leu, L_Ile, L-His, L_Lys and Dipeptide consisting of one amino acid selected from L_Arg

Group N: One amino acid selected from L-Phe, L-Tyr and L-Trp, L-Ala, L_Ser, L-Thr, Cys, Shiichi Val, Shiyu eu, Ile, Ile From _Met, _Pro, _Phe, _Tyr, _Trp, _Asp, _G1u, L-Asn, L-Gln, L-His, L_Lys, L-Arg, L_Om, L_Cit, Gly and / 3 -Ala A dipeptide consisting of one amino acid selected from L-Leu and L-Ala, L_Ser, L-Thr, L_Glu, L_As n, L_Gln, L-His, L_Lys, L_Arg and Gly. And a dipeptide consisting of L-Pro and one amino acid selected from L-His, L-Lys and L-Arg, preferably L-Phe and L-Ala, L_Ser, L-Thr, A dipeptide consisting of one amino acid selected from L-Cys, L-Phe, L-Tyr, L-Tr p, L-His, L-Lys, L_Arg and Gly, L—Trp and L—Ala, L— One amino acid selected from Ser, L-Thr, L-Cys, L-His, L-Lys, L-Arg, Gly and / 3—Ala A dipeptide consisting of one amino acid selected from L-Leu and L-Ala, L_Ser, L_Thr and Gly, and a dipeptide consisting of L-Pro and L-His, more preferably L-Phe Or a dipeptide consisting of L-Trp and one amino acid selected from L-His, L_Lys and L_Arg, and a dipeptide consisting of L-Pro and L-His Group O: One amino acid selected from L-Leu, L_Met, L-Phe, L-Tyr, L_Trp and L_His and L—Ala, L-Ser, L_Thr, L_Cys, L_Val, L-Leu, L_Ile, L- A dipeptide consisting of one amino acid selected from Met, L_Pro, L_Phe, L-Tyr, L_Trp, L_Asp, L_Glu, L_Asn, L_Gln, L_His, L_Lys, L-Arg L_Orn, L_Cit, Gly and / 3 -Ala, And a dipeptide consisting of one amino acid selected from L-Lys, L-His and L-Arg and L-Cys, preferably LL eu and L—Ala, L—Ser, L—Thr, L-Cys, L — A dipeptide consisting of one amino acid selected from Met, L—Phe, L—Tyr, L—Trp, L—Glu, L—Asn, L-Gin, L-Cit, Gly and / 3 -Ala, LM et and L—Ala, L_Ser, L_Thr, L-Cys, L—Val, L-Leu, L_Ile, L-Met, L_Phe, L_Tyr, L-Trp, L—Gln, L-His, L—Lys, L- Dipep consisting of one amino acid selected from Arg, L—Cit, Gly and / 3—Ala L-Phe or L-Trp and L-Ala, L_Ser, L_Thr, L-Cys, L-Val, L_Leu, L-Ile, L-Met, L_Phe, L_Tyr, L-Trp, L_Asp, L_Asn, L_Gln, Dipeptide consisting of one amino acid selected from L-His, LL ys, L-Arg, L_Cit, Gly and / 3-Ala, L-Tyr and L-Ala, L-Ser, L-Thr, L-Cys , L-His, L-Lys, L-Arg, and Gly, a dipeptide consisting of one amino acid, and L-His and L-Ala, L_Ser, L_Thr, L-Cys, and _Val eu, Shi—Ile, Shiichi Asn, Shi _Gln, Shi _Fiis, Shi—shi ys, Shiichi Arg, Shi _Cit, Gly and / 3 _A1 Dipeptide consisting of L-Phe or L-Tyr and one amino acid selected from L-Val, L-Leu and L-Ile

Group P: L_Phe, L-Trp or L-Tyr and L_Phe, L-Tyr, L-Trp, L_Asp and L_Gln, one dipeptide consisting of one amino acid, and L-Gln and L_Phe, L-Trp, L A dipeptide consisting of one amino acid selected from -Tyr, L_Glu, L-His, L-Lys and L-Arg, preferably selected from L-Gln and L_Phe, L-Trp, L_Tyr and L_Glu 1 Dipeptides composed of various amino acids, more preferably dipeptides composed of L-Phe or L-Tyr and L_Gln, and dipeptides composed of L-Glu and L-Gln

Group Q: Gly or L-Ala and a dipeptide consisting of one amino acid selected from L_Met, L_Pro, L_Phe, L_Tyr and L_Trp, a dipeptide consisting of L-Ser and L-Gln or L-Orn, L_Thr and L Dipeptide consisting of one amino acid selected from -Glu, L-His, L-Lys, L-Arg, L_Cit and / 3-Ala, L-Cys and L-Cys, L_Val, L-Leu, L_Ile, L_Met , L-Pro, L-Phe, L-Tyr, L-Trp, L-Asp, L-Glu, L-Asn, L-Gln, L-His, L-Lys, L-Arg and / 3 -Ala A dipeptide consisting of one selected amino acid, a dipeptide consisting of one amino acid selected from L-Glu and L-Val, L_Leu and L_Ile, selected from L-Ile and L_Phe, L-Tyr, L_Trp and L_Cit A dipeptide consisting of one amino acid selected from the group consisting of L-Pro and L_Phe, L-Tyr, L-Trp, L-His, L_Lys, L_Arg, L_Om, L_Cit and / 3-Ala. One amino acid selected from dipeptides, L_Phe, L-Tyr and L-Trp and one amino acid selected from L_Phe, LT yr, L-Trp, L_Asp, L-Gln, L-His, L_Lys and L_Arg A dipeptide consisting of one amino acid selected from L-Gln and L_His, L_Lys and L_Arg, a dipeptide consisting only of β-Ala, and a dipeptide consisting only of L-Cit, preferably Gly or L- A dipeptide consisting of Ala and L-Pro, a dipep consisting of L_Cys and L-Glu And a dipeptide consisting of L-Pro and one amino acid selected from L-His, L-Lys and L-Arg, more preferably selected from L-Pro and L-His, L-Lys and L-Arg. A dipeptide consisting of a single amino acid

(2) Dipeptide production method using culture of microorganism or transformant or treated product of culture as enzyme source

As the culture of the microorganism or transformant used as the enzyme source in the production method of the present invention, the culture obtained by culturing the microorganism or transformant by the culture method described above can be used. The treated product of the culture of the microorganism or transformant includes a concentrate of the culture, a dried product of the culture, a cell obtained by centrifuging or filtering the culture, As an enzyme source such as a dried product, a freeze-dried product of the cell, a surfactant-treated product of the cell, a solvent-treated product of the cell, an enzyme-treated product of the cell, and an immobilized product of the cell Including viable cells that retain the same functions as microorganisms !, and those obtained from ultrasonically treated products of the cells, mechanically ground products of the cells, and the treated cells. Crude enzyme extract.

When using a transformant or a culture of microorganisms or a processed product of the microorganism as an enzyme source, one or more amino acids used as a substrate can be increased by the same power S as the above (1). .

The amount of the enzyme source varies depending on the specific activity of the enzyme source, etc. For example, 5 to 1000 mg, preferably 10 to 400 mg, is added as wet cell weight per 1 mg of amino acid used as a substrate. [0072] The amino acid used as the substrate can be added to the aqueous medium in the same manner as in the above (1). As in (1) above, ATP can be present in an aqueous medium and used as an energy source.

As the aqueous medium, the medium described in (1) above can be used. In addition, the culture supernatant of the culture of the microorganism or transformant used as the enzyme source can also be used as the aqueous medium.

[0073] In the above production method, if necessary, a surfactant or an organic solvent may be added to the aqueous medium. Surfactants include nonionic surfactants such as polyoxyethylene 'octadecylamine (eg, Niimine S_215, manufactured by NOF Corporation), cetyltrimethyl ammonium bromide, and alkyldimethyl benzyl ammonium chloride (eg, cation F2-40E). Cationic surfactants such as Nippon Oil & Fats Co., Ltd., anionic surfactants such as lauroyl 'zalcosinate, and tertiary amines such as alkyldimethylamine (eg, tertiary amine FB, manufactured by Nippon Oil & Fats Co., Ltd.) Any one can be used as long as it promotes the production of a galactose-containing complex carbohydrate. The surfactant is usually used at a concentration of 0.1 to 50 g / 1. Examples of the organic solvent include xylene, toluene, aliphatic alcohol, acetone, ethyl acetate and the like, and are usually used at a concentration of 0.1 to 50 ml / 1.

[0074] The reaction conditions for the dipeptide production reaction may be the same as in (1) above.

Yes

As the dipeptide produced by the above method, the same dipeptide as in the above (1) can be mentioned.

In the production methods (1) and (2) above, the dipeptide produced and accumulated in the aqueous medium can be collected by a normal method using activated carbon, ion exchange resin or the like, or extraction, crystallization, thinning with an organic solvent, etc. It can be carried out by layer chromatography, high performance liquid chromatography or the like.

Examples are shown below, but the present invention is not limited to the following examples.

Example 1

[0076] Construction of a dipeptide synthase expression strain

(1) Dipeptide synthase expression strain derived from Streptococcus mutans ATCC25175 strain Creation

A DNA fragment having the base sequence represented by SEQ ID NO: 6 was obtained from the chromosomal DNA of Streptococcus mutans ATCC25175 strain as follows.

[0077] Streptococcus mutans ATCC25175 strain was applied to BL agar medium [58g / l BL agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), using three 8.5cm diameter petri dishes] and incubated at 30 ° C for 2 days did.

The cells grown on the three petri dishes are suspended in 3 ml of sterile deionized water using disposable sponges, and collected from the cells collected by centrifugation, UltraClean (TM) Microbial DNA Isolation Kit (Emovery) Chromosomal DNA was prepared using a laboratory company.

[0078] Synthetic DNA having the nucleotide sequences represented by SEQ ID NOs: 11 and 12 was used as a primer set, and PCR was performed using the chromosomal DNA of Streptococcus mutans ATCC25175 strain as a saddle type. PCR was performed with 0 · l _ί g of chromosomal DNA, 0.3 〃mol / 1 primer, 1 nits of KOD plus DNA polymerase (Toyobo), 5 ^ L of KOD plus DNA polymerase X 10 buffer (Toyobo) ), 200 mol / 1 each of dNTP (dATP, dGTP, dCTP and dTTP), lmmol / 1 MgSO. 50 L was prepared, heated at 94 ° C. for 2 minutes,

Four

Repeat the process for 15 seconds at ° C, 30 seconds at 55 ° C, 1 minute 40 seconds at 68 ° C 30 times, and then at 68 ° C.

This was done by warming for 2 minutes.

[0079] 1/10 volume of the reaction solution was subjected to agarose gel electrophoresis, and it was confirmed that a DNA fragment of about 1.2 kb was amplified. The DNA fragment was purified from the remaining reaction solution using PureLink ™ PCR Purification kit (Invitrogen) and dissolved in 50 1 of sterile ultrapure water.

Next, using the DNA solution 101 obtained above, the DNA fragment was cleaved with a restriction enzyme and then subjected to agarose gel electrophoresis, and about 1.2 kb was obtained using PureLink (TM) PCR Purificat ion kit. DNA fragments were recovered.

[0080] After 0.5 μg of the expression vector pColdl (Takara Bio) was digested with restriction enzymes ^ m ^ I and ϋίΙΙΙ, the digestion by restriction enzyme was confirmed by agarose gel electrophoresis. An approximately 4.4 kb DNA fragment was recovered.

The approximately 1.2 kb DNA fragment and the approximately 4.4 kb DNA fragment obtained above were reacted at 16 ° C for 4 hours using a ligation kit (Takara Bio Inc.) to obtain recombinant DNA.

[0081] Using the recombinant DNA, Escherichia coli DH5a strain (Toyobo Co., Ltd.) After transformation by a method using ions [Proc. Natl. Acad. ScL, USA, 69, 2110 (1972)], the transformant was applied to an LB agar medium containing 100 g / ml ampicillin, and 3 Incubated overnight at 0 ° C.

The grown colonies of the transformant were separated from the PCR reaction solution [0.4 H mol / 1 synthetic DNA having the nucleotide sequence represented by SEQ ID NO: 12, 0.4 01 ₀ 1/1 (： 01 (1-? Primer ( Takara Bio Inc.), lunit Z-taq (Takara Bio Inc.), 2 1 ΙΟχΖ-taq Buffer (Takara Bio Inc.), 200 01 ₀ 1/1 (^ Ding ?? dGTP, dCTP and dTTP)] and PCR was performed.

PCT is heated at 96 ° C for 2 minutes, and then the process of 96 ° C for 10 seconds, 50 ° C for 10 seconds, 72 ° C for 50 seconds is repeated 30 times, and further heated at 72 ° C for 5 minutes. It was done by doing. It was confirmed by agarose gel electrophoresis that a DNA fragment of about 1.2 kb was amplified.

[0082] The nucleotide sequence of the DNA fragment was determined by a known method and confirmed to have the nucleotide sequence represented by SEQ ID NO: 6.

Based on the above, it was confirmed that the transformant had a plasmid in which an approximately 1.2 kb DNA fragment having the base sequence represented by SEQ ID NO: 6 was ligated downstream of the SSEA promoter of pColdl.

[0083] The transformed strain was cultured overnight in LB medium containing 100,1 g / ml ampicillin, and a plasmid was prepared from the obtained culture solution by the alkaline SDS method (Molecular 'Cloning 3rd Edition). This plasmid was named pSMU1321c.

After transforming Escherichia coli BL21 (DE3) strain (manufactured by Novagen) with pSMU1321c by a method using calcium ion, the transformant was applied to an LB agar medium containing 100 g / ml ampicillin. The transformant was selected by culturing overnight at 30 ° C.

[0084] The transformant was named Escherichia coli BL21 (DE3) / pSMU1321c.

(2) Acquisition of Streptococcus pneumoniae ATCC BAA-334-derived dipeptide synthase expression strain

From the chromosomal DNA of Streptococcus pneumoniae ATCC BAA-334 strain, a DNA fragment having the base sequence represented by SEQ ID NO: 7 was obtained as follows.

[0085] A synthetic DNA having the nucleotide sequence represented by SEQ ID NOs: 13 and 14 was used as a primer set, and the chromosomal DNA of Streptococcus pneumoniae ATCC BAA-334 strain (American An Type Culture Collection (hereinafter abbreviated as ATCC) was used for PCR. PCR was performed under the same reaction conditions by preparing a reaction solution having the same composition as in (1) above. After confirming that a DNA fragment of approximately 1.2 kb was amplified by agarose gel electrophoresis, purify the DNA fragment from the reaction mixture using PureLink (TM) PCR Purification Kit, and sterilize it in 50 111 1 sterile ultrapure water. Dissolved.

[0086] Next, using 10 μ1 of the DNA solution obtained above, the DNA fragment was cleaved with EmEi and Mindni, then subjected to agarose gel electrophoresis, and about 1.2 kb using the PureLink ™ PCR Purification kit. DNA fragments were recovered.

After cleaving 0.5 g of pCoidi with E ^ Ei and m, and performing agarose gel electrophoresis to confirm cleavage by the restriction enzyme, a DNA fragment of about 4.4 kb was recovered by the same method as described above.

[0087] The about 1.2 kb DNA fragment and the about 4.4 kb DNA fragment obtained above were reacted at 16 ° C for 4 hours using a ligation kit to obtain recombinant DNA.

Using the recombinant DNA, Escherichia coli DH5a strain was transformed by a method using calcium ions, and the transformant was applied to LB agar medium containing 100 g / ml ampicillin, and then 30 ° C.晚 -cultured.

[0088] A colony of the grown transformant was transformed into a PCR reaction solution [0.4 mol / 1 synthetic DNA having the nucleotide sequence represented by SEQ ID NO: 14, 0.4 0101/1 pCold-F Primer, lunit Z_taq

, 2 μ 1 10xZ-taq Buffer, 200 μmol / 1 dNTP (dATP, dGTP, dCTP and dTTP)], and perform PCR reaction under the same conditions as in (1). It was confirmed that a 1.2 kb DNA fragment was amplified! /.

[0089] The base sequence of the DNA fragment was determined by a known method, and confirmed to have the base sequence represented by SEQ ID NO: 7.

Based on the above, it was confirmed that the transformant had a plasmid in which an approximately 1.2 kb DNA fragment having the nucleotide sequence represented by SEQ ID NO: 7 was linked downstream of the pColdl promoter.

[0090] The transformed strain was cultured overnight in LB medium containing 100,1 g / ml ampicillin, and a plasmid was prepared from the obtained culture solution by the alkaline SDS method. The resulting plasmid was designated as pSP0885. Named.

After transforming Escherichia coli BL21 (DE3) strain with pSP0885 by a method using calcium ions, the transformant was applied to LB agar medium containing 100 g / ml ampicillin, and then incubated at 30 ° C. After overnight culture, transformants were selected.

[0091] The transformed strain was named Escherichia coli BL21 (DE3) / pSP0885.

(3) Acquisition of dipeptide synthase expression strain derived from Actinobacillus pleuroneumonie ATCC 27088

A DNA fragment having the nucleotide sequence represented by SEQ ID NO: 8 was obtained from the chromosomal DNA of the Lactobacillus pleuroneumonier ATCC 27088 strain (available from ATCC) as follows.

[0092] PCR was performed using the synthetic DNA having the nucleotide sequences represented by SEQ ID NOs: 15 and 16 as a primer set, and using the chromosomal DNA of Actinobacillus' Pluroneumonie ATCC 27088 strain as a base. PCR was performed under the same reaction conditions by preparing a reaction solution having the same composition as in (1) above.

After confirming that a DNA fragment of approximately 1.2 kb was amplified by agarose gel electrophoresis, purify the DNA fragment from the reaction mixture using PureLink (TM) PCR Purification Kit, and sterilize it in 50 111 1 sterile ultrapure water. Dissolved.

[0093] Next, using 10 μ1 of the DNA solution obtained above, the DNA fragment was cleaved with EmsHI and HindlH, followed by agarose gel electrophoresis, and about 1.2 kb using PureLink ™ PCR Purification Kit. DNA fragments were recovered.

After cleaving 0.5 g of pCoidi with ^ mMi and Minm and performing agarose gel electrophoresis to confirm cleavage with restriction enzymes, a DNA fragment of about 4.4 kb was recovered by the same method as described above.

[0094] The about 1.2-kb DNA fragment and the about 4.4-kb DNA fragment obtained above were reacted at 16 ° C for 4 hours using a ligation kit to obtain recombinant DNA.

Using the recombinant DNA, Escherichia coli DH5a strain was transformed by a method using calcium ions, and the transformant was applied to LB agar medium containing 100 g / ml ampicillin, and then 30 ° C.晚 -cultured. [0095] Colonies of the grown transformant were transformed into a PCR reaction solution [0.4 mol / 1 synthetic DNA having the nucleotide sequence represented by SEQ ID NO: 16, 0.4 0101/1 pCold-F Primer, lunit Z_taq, Suspend in 2 μ 1 10xZ-taq Buffer, 200 μmol / 1 dNTP (dATP, dGTP, dCTP and dTTP)], perform a PCR reaction under the same conditions as in (1), and perform approximately 1.2 times by agarose gel electrophoresis. It was confirmed that the kb DNA fragment was amplified! /.

[0096] The base sequence of the DNA fragment was determined by a known method and confirmed to have the base sequence represented by SEQ ID NO: 8.

Based on the above, it was confirmed that the transformant had a plasmid in which a DNA fragment of about 1.2 kb having the base sequence represented by SEQ ID NO: 8 was ligated downstream of the pColdl promoter.

The transformed strain was cultured overnight in LB medium containing 100,1 g / ml ampicillin, a plasmid was prepared from the obtained culture broth by the alkaline SDS method, and the resulting plasmid was named pAple0835.

After transforming Escherichia coli BL21 (DE3) strain with pAple0835 by a method using calcium ions, the transformant was applied to LB agar medium containing 100 g / ml ampicillin, and then 30 ° C. And transformed overnight.

The transformed strain was named Escherichia coli 'BL21 (DE3) / pAple0835.

(4) Acquisition of a dipeptide synthase expression strain derived from Photolabs 'Luminescence DSM15139 strain Base from SEQ ID NO: 9 from the chromosomal DNA of Photolabs' Noreminesense DSM15139 strain (DSM: available from Deutsche Sammlung von Microorg anism und Zellkulturen GmbH) A DNA fragment having the sequence was obtained as follows.

[0099] First, from Photolabs' Luminescence DSM15139 strain, chromosome D was prepared in the same manner as (1).

NA was prepared.

Synthetic DNA having the nucleotide sequences represented by SEQ ID NOs: 17 and 18 was used as a primer set, and PCR was carried out using the chromosomal DNA of Photolabs luminescence DSM15139 strain as a saddle type. PCR was carried out under the same reaction conditions by preparing a reaction solution having the same composition as in (1) above.

[0100] Agarose gel electrophoresis confirmed that a DNA fragment of about 1.5 kb was amplified. Thereafter, the DNA fragment was purified from the reaction solution using a PureLink ™ PCR Purification kit and dissolved in 50 111 sterilized ultrapure water.

Next, using the DNA solution 101 obtained above, the DNA fragment was cleaved with ^ m ^ I and πίπ ^ ιιι and then subjected to agarose gel electrophoresis, and about 1.5 using PureLink (TM) PCR Purification kit. A kb DNA fragment was recovered.

[0101] After 0.5 μg of pColdl was cleaved with ^ mHI and aliquot, agarose gel electrophoresis was performed to confirm cleavage by restriction enzymes, and then a DNA fragment of about 4.4 kb was recovered by the same method as described above.

The approximately 1.5 kb DNA fragment and the approximately 4.4 kb DNA fragment obtained above were reacted at 16 ° C. for 4 hours using a ligation kit to obtain recombinant DNA.

[0102] A colony of the grown transformant was transformed into a PCR reaction solution [0.4 mol / 1 synthetic DNA having the nucleotide sequence represented by SEQ ID NO: 18, 0.4 0101/1 pCold-F Primer, lunit Z_taq, Suspend in 2 μ 1 10xZ-taq Buffer, 200 μmol / 1 dNTP (dATP, dGTP, dCTP and dTTP)], perform a PCR reaction under the same conditions as in (1), and perform about 1.5 times by agarose gel electrophoresis. It was confirmed that the kb DNA fragment was amplified! /.

[0103] The base sequence of the DNA fragment was determined by a known method, and confirmed to have the base sequence represented by SEQ ID NO: 9.

Based on the above, it was confirmed that the transformant retained the plasmid with an approximately 1.5 kb DNA fragment ligated downstream of the promoter of pColdl! /.

The transformed strain was cultured overnight in LB medium containing 100 g / ml ampicillin, and a plasmid was prepared from the obtained culture solution by the alkaline SDS method. The resulting plasmid was named pPlu 218.

[0104] Escherichia coli BL21 (DE3) strain was transformed with pPlu 218 by a method using calcium ions, and the transformant was applied to an LB agar medium containing 100 g / ml ampicillin. The transformant was selected by culturing overnight at 30 ° C. The transformant was named Escherichia coli BL21 (DE3) / pPlul218.

(5) Construction of a dipeptide synthetase expression strain derived from Treponema dentikola ATCC 35405

A DNA fragment having the nucleotide sequence represented by SEQ ID NO: 10 was obtained from the chromosomal DNA of Treponema denticola ATCC 35405 as follows.

[0105] Synthetic DNA having the nucleotide sequences represented by SEQ ID NOs: 19 and 20 was used as a primer set, and the chromosomal DNA of Treponema 'Denticola ATCC 35405 (ATC

PCR was performed using C). For PCR, prepare a reaction solution with the same composition as (1).

The reaction was performed under the same reaction conditions as in (1).

After confirming that a DNA fragment of about 1.6 kb was amplified by agarose gel electrophoresis, the DNA fragment was recovered from the reaction solution and dissolved in 50 1 sterile ultrapure water.

[0106] Next, using the DNA solution 10H1 obtained above, the DNA fragment was cleaved with EmsHI and HindlH and then subjected to agarose gel electrophoresis. As in (1), a 1.6 kb DNA fragment was recovered. It was.

After cleaving 0.5 g of pColdi with Emsili and Hindiii and performing agarose gel electrophoresis to confirm cleavage with restriction enzymes, a DNA fragment of about 4.4 kb was recovered by the same method as described above.

The approximately 1.6 kb DNA fragment and the approximately 4.4 kb DNA fragment obtained above were reacted at 16 ° C. for 4 hours using a ligation kit to obtain recombinant DNA.

[0107] After the Escherichia coli DH5 α strain was transformed with the recombinant DNA by a method using calcium ions, the transformant was applied to an LB agar medium containing 100 g / ml ampicillin, Cultivation was performed at 30 ° C.

Colonies of transformants have been grown, the synthetic DNA having the nucleotide sequence represented by SEQ ID NO: 20 of the PCR reaction solution [0.4 H mol / 1, 0. 4 0101/1 of (:? 01 (1- Primer, lunit Z_taq, 2 μ 1 10xZ-taq Buffer, 200 μmol / 1 dNTP (dATP, dGTP, dCTP, and dTTP)], suspended in the same conditions as in (1), and agarose gel electrophoresis Confirmed that a DNA fragment of about 1.2 kb was amplified! /.

[0108] The nucleotide sequence of the DNA fragment was determined by a known method, and the nucleotide sequence represented by SEQ ID NO: 10 It was confirmed to have

From the above, it was confirmed that the transformant had a plasmid fragment of about 1.6 kb ligated downstream of the promoter of pColdl!

The transformed strain was cultured overnight in an LB medium containing 100 g / ml ampicillin, a plasmid was prepared from the obtained culture solution by the alkaline SDS method, and the resulting plasmid was named pTDE2209.

[0109] Escherichia coli BL21 (DE3) strain (manufactured by Novagen) was transformed with pTDE2209 by a method using calcium ions, and the transformant was applied to an LB agar medium containing 100 g / ml ampicillin. After that, the transformant was selected by culturing overnight at 30 ° C.

The transformed strain was named Escherichia coli BL21 (DE3) / pTDE2209.

Example 2

[0110] Production of protein having dipeptide synthesis activity

Escherichia coli obtained in Example 1 BL21 (DE3) / pSMU1321c, BL21 (DE3) / pSP088 5, BL21 (DE3) / pAple0835, BL21 (DE3) / pPlul218, BL21 (DE3) / pTDE2209 100 The tube was inoculated into a test tube containing 10 ml of LB medium containing g / ml of ampicillin and cultured at 30 ° C with shaking. Of the resulting culture broth, 500 1 was inoculated into a 200 ml Erlenmeyer flask containing 50 ml of LB medium each containing 100 g / ml ampicillin. After shaking culture at 30 ° C for 3 hours, add isopropyl- / 3-D-thiogalatatopyranoside (IPTG) to each flask to a final concentration of 0.4 mmol / l, and further shake culture at 15 ° C overnight. did. The culture solution was centrifuged to obtain bacterial cells.

[0111] The protein with the His tag added was purified from the supernatant obtained by crushing the wet cells by sonication and then centrifuged, using HisTrap (manufactured by Amersham).

Example 3

[0112] Production of dipeptides using His-tagged proteins

(1) Dipeptide production using Streptococcus mutans-derived dipeptide synthase In Example 2, the His-tagged protein obtained using Escherichia coli BL21 (DE3) / pSMU1321c was added at 100 g / l, lOOmmol / 1 Tris-HCl buffer (ρΗ8 · 0), 20 mmol / l magnesium sulfate, 20 mmol / l ATP, 20 mmol / l L-Ala, L-Gln, L-Glu, L_Val, LL eu, L—Ile, L—Pro, L—Phe, L—Trp, L—Met, L—Ser, L—Thr, L—Cys, L—Asn, L—Tyr, L—Lys, L-Arg, Two amino acids selected from L_His, L-Asp, Gly, β-Ala and L_Cit or 40 mmol / l — Ala, then _Gln, then — Glu, then — Val, then — eu, then _Ile, _Pro, _Phe, Trp, L—Met, L—Ser, L—Thr, L—Cys, L—Asn, L—Tyr, L—Lys, L—Arg, L—His, L—Asp, A reaction solution consisting of one kind of amino acid selected from Gly, / 3-Ala and L-Cit was prepared and reacted at 30 ° C for 24 hours. After completion of the reaction, the amount of phosphoric acid liberated in the reaction solution was quantified using Detamina L IP (manufactured by Kyowa Medettas), CE / MS (Capillary Electrophoresis Mass Spectrometry) analysis and FMOC (fluorenylmethyl chloroformate) derivatization reaction The products were analyzed by three methods of HPLC analysis using

[0113] For CE-MS analysis, an Agilent CE / MS system (manufactured by Agilent Technologies) was used as a measuring device. For capillary electrophoresis, a quartz glass capillary with a length of 100 cm and an inner diameter of 50 m was used, and electrophoresis was performed at 20 ° C and a voltage of 30 kV using 1 mol / 1 formic acid as a buffer solution.

In mass spectrometry, the capillary voltage was 3500 V and nitrogen gas at 300 ° C was used as the dry gas. The analysis was performed under conditions where molecular weight from 70 to 500 was detected using 5 mmol / 1 ammonium acetate (dissolved in 50% methanol) as a sheath liquid and a flow rate of 10 to 1 / min.

[0114] FMOC derivatization was carried out by adding 49 μ 1 0 · lmol / 1 borate buffer (adjusted to ρΗ9 · 0 with sodium hydroxide) to reaction solution 1 [1 1 and mixing 50 1 1.5 A mg / ml FMOC acetone solution was added and reacted at room temperature for 30 minutes. After completion of the reaction, add 500 1 25% (v / V) acetonitrile solution (20 mmol / 1 L-proline, ρΗ8.0 · 0.25 mol / l borate buffer) to the reaction mixture, and sample for HPLC analysis It was. The HPLC analysis was basically performed under the following conditions, and the pH and concentration gradient schedule of Solution A described below were slightly changed depending on the dipeptide to be detected.

[0115] Develosil ODS-HG-5 (manufactured by Nomura Chemical Co., Ltd.) was used as the separation column, the column temperature was 40 ° C, and the flow rate was 1.0 ml / min.

The moving bed uses solution A [20mmol / 1 ammonium hydrogenphosphate solution (adjusted to pH 5.0 with aqueous ammonia): methanol = 85:15] and solution B (acetonitrile: water = 9: 1) from the start of analysis. Up to 2 minutes, solution A: Solution B = 75:25, 2 to 21 minutes, solution when 21 minutes are reached The ratio of solution B is increased linearly so that A: solution B = 55: 45, and solution A: solution B = 45: 55 is linear when it becomes 36 minutes from 21 to 36 minutes. Increase the ratio of solution B to 36 to 37 minutes, and increase the ratio of solution B linearly so that solution A: solution B = 1:99 when 37 minutes are reached, and from 37 to 39 minutes Solution A: Solution B = l: Hold at 99, and from 39 to 44 minutes, decrease the ratio of Solution B linearly so that Solution A: Solution B = 72: 25 at 44 minutes. From 44 to 50 minutes, solution A: solution B = 75: 25.

[0116] The detection of the dipeptide was performed by detecting the emission of 606 nm by the excitation light of 254 nm.

As a result, in the method based on the quantification of liberated phosphate,

(a) L_Phe and L—Ala, L_Ser, L_Thr, L_Cys, L—Val, L-Leu, L_Ile, L-Met, L_Pro, L-Phe, L_Tyr, L_Trp, L_Asp, L_Glu, L_Asn, L_Gln, L_His, L -Lys, L_Arg, L_Om, L-Cit, Gly or 0 -Ala linked dipeptide,

(b) L_Tyr and L—Ala, L_Ser, L_Thr, L_Cys, L—Val, L_Pro, L_Tyr, L_Trp, L—Asp, L-Glu, L-Asn, L-Gln, L-His, L-Lys, L_Arg , L-Orn, L-Cit, Gly or β-Ala linked dipeptide,

(c) L_Trp and L—Ala, L_Ser, L_Thr, L_Cys, L—Val, L-Leu, L_Ile, L-Met, L_Pro, L-Trp, L—Asp, L—Glu, L—Asn, L—Gln L-His, L-Lys, L-Arg, L-Om, L-Cit, Gly or β-Ala-bound dipeptide,

(d) L_His and L_Ala, L_Ser, L_Thr, L_Cys, L_Val, L-Leu, L-Met, L_Pro, L-Asp, L-Glu, L-Asn, L-Gln, L-His, L-Lys, L_Arg , L-Orn, L-Cit, Gly or β-Ala-bound dipeptide, and

(e) a dipeptide in which L-Leu and L-Cys are bound,

Was found to have generated.

[0117] Also, in CE-MS analysis,

(£) _? 1 ₁ 6 and -8 1 &, L-Cys, L-VaU L-Met, L_Phe, L-Trp, L_Gln, L_His, L-Lys, L-Arg, Gly or β-Ala Dipeptide,

(g) a dipeptide in which L-Tyr and L-Cys or Gly are bound,

(h) Dipeptide containing L-Trp and L-Ala, L-Cys, L-Leu, L_Met, L_His or / 3-Ala De ...

(1) Dipeptides with L-His and L-Ala, L-Ser, L-Cys, L-Met, L-Pro, L-Gln, L-His, Gly or β-Ala

(j) a dipeptide in which L-Leu and L-Cys are bound,

That was the power that was generated.

[0118] Further, HPLC analysis showed that 19.6 mol / 1 L-Phe-L-Val and 31.5 mol / 1 L_Phe-L-Arg were produced.

(2) Dipeptide production using Streptococcus pneumoniae-derived dipeptide synthase

In Example 2, using the His-tagged protein obtained using BL21 (DE3) / pSP0885, a dipeptide production reaction was performed under the same conditions as in (1).

[0119] As a result, in the method based on the determination of free phosphoric acid,

(b) L_Trp and L—Ala, L_Ser, L_Thr, L_Cys, L—Val, L-Leu, L_Ile, L-Met, L_Pro, L-Tyr, L_Trp, L_Asp, L_Glu, L_Asn, L_Gln, L_His, L_Lys, L_Arg , L_Orn, L_Cit, Gly or 0-Ala linked dipeptide,

(c) a dipeptide in which L-Leu and L-Ser, L-Glu, L_Asn, L-Gln, L_His or Gly are bound,

(d) a dipeptide in which L-Tyr and L_Asn, L_Gln or L_His are bound, and

(e) a dipeptide in which L-His and L-Pro are bound,

That was the power that was generated.

[0120] Also, in CE-MS analysis,

(f) Dipeptide in which L-Phe and L-Cys, L_Phe, L-His, L_Arg or Gly are bound,

(g) a dipeptide in which L-Trp and L-Ala, L-Cys, L-Arg, Gly or β-Ala are bound,

(h) a dipeptide in which L-Leu and Gly are bound, and

(i) a dipeptide in which L-His and L-Pro are bound,

That was the power that was generated. [0121] Furthermore, HPLC analysis revealed that 0 · 12 μmol / 1 L-Pro-L-His and 1 · 24 μmol / 1 L-Phe-L-Arg were produced.

(3) Dipeptide production using Dipeptide synthase derived from Actinobacillus pleuroneumonier

In Example 2, using a His-tagged protein obtained using BL21 (DE3) / pAple0835, a dipeptide production reaction was performed under the same conditions as in (1).

[0122] As a result, in the method based on the determination of free phosphoric acid,

(a) L_Leu and L_Ser, L_Cys, L-Met, L_Phe, L_Tyr, L-Trp, L_Glu, L_Asn, L_Gln, L-His, L-Lys, L-Arg, L_Om, L_Cit, Gly or β-Ala Dipeptides,

(b) L_Met and L—Ala, L_Ser, L_Thr, L_Cys, L—Val, L_Ile, L-Met, L_Pro, L_Phe, L-Tyr, L-Trp, L-Asp, L_Glu, L_Asn, L_Gln, L_His, L -Dipeptides with Arg, L_Orn, L_Cit, Gly or β-Ala,

(c) L_Phe and L—Ala, L_Ser, L_Thr, L_Cys, L—Val, L_Ile, L_Pro, L_Phe, L_Tyr, LT rp, L—Asp, L—Glu, L—Asn, L—Gln, L—His, L—Lys, L—Arg, L—Orn, L—Cit, Gly or β-Ala-bound dipeptide,

(d) L_Tyr and L—Ala, L_Ser, L_Thr, L_Cys, L—Val, L_Pro, L_Tyr, L_Trp, L—Asp, L-Glu, L-Asn, L-Gln, L-His, L-Lys, L_Arg , L-Orn, L-Cit, Gly or β-Ala linked dipeptide,

(e) L_Trp and L—Ala, L_Ser, L_Thr, L_Cys, L_Val, L_Ile, L_Pro, L-Trp, L-Asp, L-Glu, L-Asn, L-Gln, L-His, L-Lys, L_Arg , L-Orn, L-Cit, Gly or β-Ala linked dipeptide,

) _ ^ ¾3 and -8 1 &, L-Ser, L-Thr, L_Cys, L—Val, L_Pro, L-Asp, L_Glu, L—Asn, L-Gln, L-His, L-Lys, L_Arg, L_Om , L_Cit, Gly or β-Ala-bound dipeptide and

(g) a dipeptide in which L-Lys and L-Cys are bound,

That was the power that was generated.

[0123] Also, in CE-MS analysis,

(h) L_Leu and L_Cys, L-Met, L-Trp, L_Glu, L-Asn, L_Gln, L_Cit, Gly or β-Ala Dipeptide to which is bound,

(i) a dipeptide in which L_Met and L_Cys, L_Val, L—Ile, L-Met, L_Phe, L_Tyr, L-Trp, L_Gln, L_His, L-Arg, L-Cit, Gly or β-Ala are bound,

() _? 1 ₁ 6 and — eight 1 &, L-Ser, L-Cys, L—Val, L_Ile, L_Phe, L_Tyr, L_Trp, L—Asp, L—Asn, L-Gln, L_His, L_Lys, L_Arg, Dipeptide with L_Cit, Gly or β-Ala bound

(k) a dipeptide in which L-Tyr and L_Ala, L-Cys, L_Arg or Gly are bound,

(1) L_Trp and L—Ala, L_Ser, L_Cys, L—Val, L_Ile, L-Trp, L—Asn, L_Gln, L_His, L-A rg, L-Cit, Gly or β-Ala dipeptide

(m) Dipeptide of L_His and L_Ala, L_Ser, L_Cys, L_Val, L—Asn, L_Gln, L_His, L-Lys, L—Arg, L-Cit, Gly or 0-Ala,

That was the power that was generated.

[0124] Furthermore, HPLC analysis showed that 92.5 mol / 1 L-Phe-L-Val was produced.

(4) Photolabs' dipeptide production using luminescence-derived dipeptide synthase Using the His-tagged protein obtained using BL21 (DE3) / pPlul218 in Example 2,

Dipeptide production reaction was performed under the same conditions as in (1).

As a result, in the method based on the quantification of liberated phosphate,

(a) a dipeptide in which L-Tyr and L_Tyr, L_Trp, L_Asp or L_Gln bind

(b) It was found that a dipeptide in which L-Gln and L-Phe, L_Glu or L_Arg were bound was produced.

[0125] In addition, CE-MS analysis revealed that a dipeptide in which L-Gln and L-Phe or L-Glu were bound was produced.

Furthermore, HPLC analysis shows 4.00 01 ₀ 1/1 and-? 1 ₁ 6- and -01. 0.06 mol / 1 L-Glu-L-Gln and 0.10 a mol / 1 L-Gln-L-Glu were produced.

(5) Using dipeptide synthase derived from Treponema 'dentica, dipeptide production Using the His-tagged protein obtained using BL21 (DE3) / pTDE2209 in Example 2.

The dipeptide production reaction was performed under the same conditions as in (1).

[0126] As a result, in the method based on the determination of free phosphoric acid, (a) a dipeptide in which Gly and L-Met, L-Pro or L_Tyr are bound,

(b) a dipeptide in which L-Ser and L-Gln or Shi-Orn are bound,

(c) a dipeptide in which L-Thr and L-Glu, L-Lys, L_Cit or β-Ala are bound,

(d) then _Cys, then _Cys, then one Val, then --eu, then Ile, then _Met, then _Pro, then _Phe, then _Tyr, then --Trp ゝ —Asp, L-Glu, L-Asn, L-Gln, L_His or β-Ala-bound dipeptide,

(e) L-Leu-Glu linked dipeptide,

(f) a dipeptide in which L-Ile and L-Phe or L-Cit are bound,

(g) Dipeptide with L-Pro and L_Tyr, L-His, L-Lys, L-Orn, L_Cit or β-Ala

(h) Dipeptide in which L-Phe and L-Phe, L-Tyr, L_Trp, L_Asp, L_Gln, L_His or L_Lys are bound,

(i) a dipeptide having Tyr and L-Asp or Gin bound thereto,

(j) a dipeptide in which L-Gln and L-Arg are bound,

(k) L-Cit- L-Cit, and

(1) It was found that β-Ala-β-Ala was formed.

[0127] In CE-MS analysis,

(m) a dipeptide in which Gly and L-Pro are bound,

(n) a dipeptide in which L-Glu and L-Cys are bound, and

(o) a dipeptide in which L-Pro and L-His are bound,

Was found to have generated.

[0128] Further, in the HPLC analysis, it was found that 1 ^ ¾)-? ¾ of 0.16 01 ₀ 1/1 was produced.

As described above, the protein of the present invention has been shown to have an activity of producing various dipeptides by binding one or two amino acids by peptide bonds.

Industrial applicability

[0129] According to the present invention, a protein having an activity of synthesizing a dipeptide can be produced, and the dipeptide can be produced using the protein or a transformant or a microorganism having an ability to produce the protein.

Sequence listing free text SEQ ID NO: 11- Description of artificial sequence: Synthetic DNA SEQ ID NO: 12- Description of artificial sequence: Synthetic DNA SEQ ID NO: 13- Description of artificial sequence: Synthetic DNA SEQ ID NO: 14- Description of artificial sequence: Synthetic DNA SEQ ID NO: 15- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 16- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 17- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 18- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 19- -Description of artificial sequence: Synthetic DNA SEQ ID NO: 20- -Description of artificial sequence: Synthetic DNA

Claims

Claims [1] The protein according to any one of [1] to [3] below. [1] SEQ ID NO: protein having an amino acid sequence represented by any one of! To 5 [2] In the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, one or more amino acids are deleted, substituted or A protein comprising an added amino acid sequence and having dipeptide synthesis activity [3] An amino acid sequence having 80% or more homology with the amino acid sequence represented by any of SEQ ID NOs: 1 to 5, and a dipeptide [2] The DNA according to any one of [1] to [3] below.

[1] DNA encoding the protein according to claim 1

[2] DNA having a base sequence represented by any one of SEQ ID NOs: 6 to 10

[3] A recombinant DNA containing the DNA of claim 2.

[4] A transformant having the recombinant DNA according to claim 3.

5. The transformant according to claim 4, wherein the transformant is a transformant obtained using a microorganism as a host.

6. The transformant according to claim 5, wherein the microorganism is a microorganism belonging to the genus Escherichia.

[7] The protein according to claim 1, wherein a microorganism capable of producing the protein according to claim 1 is cultured in a medium, the protein is produced and accumulated in the culture, and the protein is collected from the culture. Manufacturing method.

[8] The production method according to claim 7, wherein the microorganism capable of producing the protein according to claim 1 is the transformant according to any one of claims 4 to 6.

[9] A culture of a microorganism having the ability to produce the protein of claim 1 or a processed product of the culture, or the protein of claim 1 and one or more amino acids in an aqueous medium, Generate and accumulate a dipeptide in the medium and collect the dipeptide from the medium A method for producing a dipeptide.

[10] The production method according to claim 9, wherein the microorganism having the ability to produce the protein according to claim 1 is the transformant according to any one of claims 4 to 6.